Deuterated cortisol (d3-cortisol) is added to a 0.1-mL urine specimen as an internal standard. Cortisol, cortisone, and d3-cortisol are extracted from the specimens using online turbulent-flow HPLC and analyzed by liquid chromatography-tandem mass spectrometry using multiple-reaction monitoring in positive mode. The following ion pairs are used for analysis: Cortisol (363.0/121.1); Cortisone (361.0/163.0); d3-Cortisol (366.0/121.2). A calibration curve, generated from stripped urine spiked standards, is included with each batch of patient specimens.(Taylor RL, Machacek DA, Singh RJ: Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 2002;48:1511-1519)